The present invention relates to protein and peptide chemistry. In particular, the present invention relates to the discovery and isolation of novel peptides whose sequences coincide with regions of the interleukin 16 (IL-16 ) receptor (CD4). The invention is also directed to the use of these novel peptides in the inhibition of IL-16-mediated biological activity.
Interleukin-16 (IL-16) was first described in 1982 as lymphocyte chemoattractant factor (LCF; Center, et al., (1982) J.Immunol. 128: 2563-2568; Cruikshank, et al., (1982) J.Immunol. 128: 2569-2574). Subsequent studies showed that IL-16 is a multifunctional cytokine, selectively inducing the migration of CD4+ T cells, eosinophils and monocytes. IL-16 also acts as a growth factor for resting CD4+ T cells, promoting their entry into the G1 phase of the cell cycle, and inducing interleukin-2 receptor and major histocompatibility (MHC) class II protein expression on the cell surface. These activating functions are associated with intracellular signals including the synthesis of inositol trisphosphate (IP3) and a transient increase of intracellular Ca2+ concentration (reviewed in Cruikshank, et al. (1998) International Reviews of Immunology 16: 523-540).
CD4 is a xcx9c55 kDa type 1 integral cell surface glycoprotein with four extracellular immunoglobulin-like extracellular domains (D1-D4), a single membrane-spanning region, and short intracytoplasmic tail which interacts with intracellular tyrosine kinases such as p56lck. The extracellular domains appear to form two rigid structures consisting of D1D2 and D3D4, with a flexible connection between D2 and D3 (Brady, et al., (1993) Science 260: 979-983). The D1D2 domain interacts with MHC class II proteins and a high affinity (Kd xcx9c10 nM) HIV-1 gp120 binding site maps to the N-terminus of the V1 region of CD4, overlapping with but distinct from the MHC class II binding site (Fleury, et al., (1991) Cell 66: 1037-1049). The seminal plasma gp17 binding site on CD4 is also located in the D1 domain, close to, but distinct from the gp120 binding site (Autiero, et al., (1997) European Journal of Biochemistry 245: 208-213).
CD4 is a receptor for IL-16. IL-16 induces chemotactic responses in CD4+ but not CD4xe2x88x92 T lymphocytes Berman, et al. (1985) 95:105-112. The T cell chemoattractant response to IL-16 is inhibited by co-incubation with Fab fragments of the anti-CD4 monoclonal antibody OKT 4, and the magnitude of the IL-16-induced cell migration by monocytes is directly proportional to the amount of CD4 expressed on the responding cells (Cruikshank,et al., (1987) J.Immunol. 138: 3817-3823). In addition, transfection of human CD4 confers IL-16-responsiveness to an otherwise unresponsive L3T4 murine hybridoma cell line as demonstrated by the induction of cell motility and rises in intracellular Ca2+ and IP3 which are inhibited by OKT4 Fab (Cruikshank, et al., (1991) J.Immunol. 146: 2928-2934).
Surface expression of CD4 is required for cells to respond to IL-16, and a direct interaction between IL-16 and CD4 was observed in co-immunoprecipitation experiments (Cruikshank, et al. (1998) International Reviews of Immunology 16: 523-540)). The CD4 ligand HIV-1 envelope glycoprotein gp120 and certain anti-CD4 antibodies mimic some of the bioactivities of IL-16 (Kornfeld, et al., (1988) Nature 335: 445-448;Ledbetter, et al., (1987) Proc.Natl.Acad.Sci.USA 84: 1384-1388; and Neudorf, et al., (1990) Cell.Immunol. 125: 301-314). Certain chemokine receptors are known to function as co-receptors with CD4 for HIV-1 infection (Feng, et al., (1996) Science 272: 872-877; Dragic, et al., (1996) Nature 381: 667-673), but it is unknown whether co-receptors are utilized by IL-16. Another soluble CD4 ligand was reported by Autiero et al. (Autiero, et al., (1991) Experimental Cell Research 197: 268-271;Autiero, et al., (1995) Eur.J.Immunol. 25:1461-1464) who isolated a human seminal plasma glycoprotein, gp17, which binds to recombinant soluble CD4 coupled to Sepharose beads as well as to CD4+ Jurkat cells. The physiological role of gp17 is presently unknown. Together, these findings indicate that CD4 is multi-functional receptor.
There is also direct physical evidence for an IL-16-CD4 interaction. IL-16 can be co-immunoprecipitated with recombinant soluble CD4, and rIL-16 partially displaces OKT4 bound to CD4 (Cruikshank, et al. (1994) Proc.Natl.Acad.Sci.USA 91: 5109-5113). Data from several laboratories indicate a high degree of sequence and functional homology for IL-16 across different animal species (Bannert, et al., (1998) Immunogenetics 47: 390-397; Keane, et al., (1998) J.Immunol. 160: 5945-5954; Leutenegger, et al., (1999) Molecular cloning and expression of feline interleukin-16, (UnPub)).
Human IL-16 induces chemotaxis of human, rat, and mouse CD4+ T cells (Center, D. M. and Cruikshank, W. W. (1982) J.Immunol. 128: 2563-2568). Murine IL-16 also induces motility and interleukin-2 receptor (IL-2R)-expression in human and murine target cells. It is therefore believed that the site(s) on CD4 interacting with IL-16 are also likely to be conserved. Comparison of the predicted amino acid sequences of CD4 across several species indicates that the D4 domain of CD4 is critical for IL-16 bioactivity.
CD4 is also the major receptor for human immunodeficiency virus-1 (HIV-1), HIV-2, and human herpes virus-7 (Dalgleish, et al., (1984) Nature 312: 763-767; Klatzmann, et al. (1984) Nature 312: 767-768; Lusso, et al., (1994) Proceedings of the National Academy of Sciences of the United States of America 91, 3872-3876). Originally identified as a differentiation antigen on T lymphocytes, CD4 was later found to be expressed on a variety cell types including monocytes, macrophages, eosinophils, hematopoietic progenitor cells, neurons, and spermatoza (Foti, et al., (1995) Journal of Laboratory and Clinical Medicine 126: 233-239). Expression of CD4 by these non-lymphocytic cells indicates that it mediates functions independent of the T cell antigen receptor, although the nature of these putative functions remain to be defined. In addition to binding MHC class II proteins, it is believed that CD4 can serve as a receptor for other soluble ligands.
Until recently, no function has been attributed to the D4 domain of CD4. Wu et al. (Wu, et al., (1997) Nature 387: 527-530) reported the x-ray crystallographic structure of recombinant soluble human D3D4 which spontaneously dimerizes at high concentration in solution. Wu et al. found that domain 4 (D4) mediates CD4 dimerization, and that the interface between dimers involves D4 domains exclusively. At the center of the interface is a pair of conserved glutamine residues (Gln345 and Gln345xe2x80x2) separated by a hydrogen-bonding distance. In their model, the level of CD4 expression when evenly distributed on a cell surface (estimated at xcx9c10xe2x88x925 M) would favor monomers. During antigen recognition, CD4 recruited by cooperative interactions at the cell-cell adhesion junction would lead to an increased local concentration favoring dimer formation. Wu et al. proposed that CD4 dimerization-mediated trans autophosphorylation is required for CD4-associated kinase activation, and subsequent intracellular signaling. In support of this model, Satoh et al. (Satoh, et al., (1996) Biochemical and Biophysical Research Communications 224: 438-443) found that D4-based peptides were capable of inhibiting a mixed lymphocyte reaction (MLR). The activity of these peptides was postulated to result from competitive binding to CD4, thus inhibiting CD4 dimerization.
Comparison of the human CD4 amino acid sequence with that of several different species revealed that immunoglobulin-like domain 4 (D4) is the most conserved extracellular region. A comparison of the amino acid sequence of the human CD4 D4 domain with the CD4 D4 domain of mice reveals that 37 out of 73 amino acids are identical. Mouse and human D4 regions have an amino acid sequence homology of approximately 63% as determined by the method of Lipman et al., (1985) Science 227: 1435-1441.
As it is established that IL-16 is a key modulator of immune and inflammatory diseases, it would be desirable to identify IL-16 antagonists, i.e., substances capable of blocking or interrupting the activity of IL-16, for use in anti-inflammatory compositions in the treatment of, e.g., asthma, rheumatoid arthritis or inflammatory bowel disease. Such compositions may also prove to be more advantageous over presently available NSAIDS, steroid based anti-inflammatory drugs and cytotoxic drugs which often have severe side effects with the continued usage that is required for chronic inflammatory diseases.
One embodiment of the present invention is directed to IL-16 antagonists.
Another embodiment of the present invention is directed to IL-16 antagonist peptides.
In accordance with the present invention, novel IL-16 antagonist peptides derived from or corresponding to the CD4 receptor have been isolated and synthesized. These peptides possess IL-16 antagonistic properties including the ability to selectively bind to IL-16 and inhibit IL-16-mediated biological activity which, for example, is associated with certain inflammatory responses in mammals. The peptides of the present invention preferably correspond to specific portions of the native human CD4 receptor and include variations thereof, and therefore are non-immunogenic when administered to humans.
The present invention also provides methods and compositions for treating IL-16 mediated disorders such as the inflammation associated with asthma, rheumatoid arthritis, inflammatory bowel disease (IBD) and systemic lupus (SLE). The present invention provides specific compositions containing at least one IL-16 antagonist peptide which inhibits, suppresses or causes the cessation of at least one IL-16-mediated biological activity in a mammal, and preferably humans.
The IL-16 antagonist peptides of the present invention are at least 4 amino acids in length and substantially correspond to the amino acids of the D4 domain of human or murine CD4 surrounding the Leu-Leu motif, i.e., L348-L349 of human CD4 D4 or L347-L348 of murine CD4 D4.
A preferred IL-16 antagonist peptide of the present invention is a tetrameric peptide having the sequence Xaa1-L-L-Xaa2, wherein Xaa1 and Xaa2 can be any amino acid.
Preferably, Xaa1 and Xaa2 are those amino acids found in the native sequence of a mammalian CD4. For example, Xaa1 can be Cys (human or murine) and Xaa2 can be Ser (human or murine). Homologs and analogs of this tetrameric peptide are also IL-16 contemplated by the present invention.
More preferably, Xaa1LLXaa2 is a tetrameric peptide identical to the native sequence of a human CD4. For example, CLLS (SEQ ID NO:2) is most preferred.
Another preferred IL-16 antagonist peptide of the present invention is a six-residue peptide having the sequence of Xaa1-Xaa2-Xaa3-Leu-Leu-Xaa4 wherein Xaa1-Xaa4, can be any amino acid (SEQ ID NO:3).
Preferably, Xaa1-4 are those amino acids found in the native sequence of a mammalian (e.g. murine and human) CD4 at the relevant positions. For example, Xaa1 can be Trp, Xaa2 can be Gln or Ala, Xaa3 can be Cys or Ala and Xaa4 can be Ser.
Even more preferably, Xaa1-Xaa2-Xaa3-L-L-Xaa4 is a 6-mer identical to the native sequence of human or murine CD4. An example of such a 6-mer includes SEQ ID NO:4 WQCLLS. Homologs and analogs of this 6-mer are also contemplated by the present invention. Examples of such homologs and analogs include: WQALLS (SEQ ID NO:5), WACLLS (SEQ ID NO:6) and WQCELS (SEQ ID NO:7).
Still another preferred IL-16 antagonist peptide of the present invention is a 6-mer having the sequence of Xaa1-Val-Xaa2-Val-Xaa3-Xaa4 wherein Xaa1-4 can be any amino acid (SEQ ID NO:8).
Preferably, Xaa1-4 are those amino acids found in the native sequence of a mammalian (e.g. murine and human) CD4 at the relevant positions. For example, Xaa1 can be Val, Xaa2 can be Gln, Xaa3 can be Val and Xaa4 can be Ala.
Even more preferably, Xaa1-Val-Xaa2-Val-Xaa3-Xaa4 is a 6-mer identical to the native sequence of human or murine CD4. An example of such a 6-mer includes SEQ ID NO:9 VVQVVA. Homologs and analogs of this 6-mer are also contemplated by the present invention. Examples of such homologs and analogs include: VKQVVA (SEQ ID NO:10) and VVQKVA (SEQ ID NO:11).
Further, according to the present invention an IL-16 antagonist peptide can be longer than a tetrameric and a 6-mer and composed of up to about 75 amino acids, as long as the antagonist peptide contains as part of the peptide, one or more of the tetrameric or 6-mer sequences described hereinabove, i.e. Xaa1LLXaa2, Xaa1-Xaa2-Xaa3-L-L-Xaa4 or Xaa1-V-Xaa2-V-Xaa3-Xaa4, and preferably Xaa1LLXaa2. Preferably, the antagonist peptide contains less than about 32 amino acids and more preferably less than about 16 amino acids.
Preferred antagonist peptides include those having sequences which coincide with the native sequence of a CD4 starting from Asn302 for human CD4, or the corresponding positions of other mammalian CD4 molecules, such as, for example Thr301 for murine CD4. Examples of such xe2x80x9clongerxe2x80x9d peptides include GMWQCLLSDSGQVLLE (SEQ ID NO:12), GMWQCLLS (SEQ ID NO:13), TGLWQCLLSEGD (SEQ ID NO:14), VSEEQKVVQVVA (SEQ ID NO:15), NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPM (SEQ ID NO:16) and TLTCEVMGPTSPKMRLTLKQENQEARVSEEQKVVQVVAPETGLWQCLLSEGDKVKMDSRIQVLSRGVNQTVF (SEQ ID NO:17).
In the amino acid sequences defined herein, the numbering of the amino acid residues corresponds to the numbering of amino acid residues in the amino acid sequence for human T-cell surface glycoprotein T4 mRNA as provided in Maddon, et al. (1985) Cell 42:93-104 (incorporated herein by reference). Homologous peptides are derived from the homologous regions of other CD4 polypeptides, such as mouse CD4, aligned in sequence for maximal homology.
In one embodiment, the amino acid sequence of the IL-16 antagonist peptide substantially corresponds to amino acids 347-350 (CLLS) of the human CD4 domain 4 (D4) (SEQ ID NO:2).
In another embodiment of the present invention, the amino acid sequence of the IL-16 antagonist peptide substantially corresponds to amino acids 343-358 (GMWQCLLSDSGQVLLE) of the human CD4 domain 4 (D4) (SEQ ID NO:12).
In still another embodiment of the present invention, the amino acid sequence of the IL-16 antagonist peptide substantially corresponds to amino acids 343-350 (GMWQCLLS) of the human CD4 domain (SEQ ID NO:13).
In another embodiment, the amino acid sequence of the IL-16 antagonist peptide substantially corresponds to amino acids 344-349 (WQCLLS) of the mouse CD4 domain 4 (D4). (SEQ ID NO:4).
In still another embodiment, the amino acid sequences of the IL-16 antagonist peptides substantially correspond to amino acid residues 333-338 of the mouse CD4 D4 region (SEQ ID NO:9).
In yet another embodiment, the amino acid sequences of the IL-16 antagonist peptides substantially correspond to amino acid residues 301-372 (TLTCEVMGPTSPKMRLTLKQENQEARVSEEQKVVQVVAPETGLWQCLLSEGDKVKMDSRIQVLSRGVNQTVF) of the mouse CD4 D4 (SEQ ID NO:17).
In still yet another embodiment, the amino acid sequences of the IL-16 antagonist peptides substantially correspond to amino acid residues 327-338 (VSEEQKVVQVVA) of the mouse CD4 D4 (SEQ ID NO:15).
In another embodiment, the amino acid sequences of the IL-16 antagonist peptides substantially correspond to amino acid residues 341-352 (TGLWQCLLSEGD) of the mouse CD4 D4 (SEQ ID NO:14).
In still yet another embodiment, the amino acid sequences of the IL-16 antagonist peptides substantially correspond to amino acid residues 302-374 (NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPM) of the human CD4 D4 (SEQ ID NO:16).
Homologs, analogs and fragments of these peptides are also contemplated by the present invention as IL-16 peptide antagonists which maintain IL-16 antagonist activity in a mammal, particularly humans.
Another aspect of the present invention provides methods of interfering with, blocking or otherwise preventing the interaction or binding of IL-16 with an IL-16 receptor by employing the IL-16 antagonists contemplated by the present invention.
The present invention also provides compositions for the treatment of IL-16-mediated disorders such as asthma, arthritis, inflammatory bowel disease (IBD), systemic lupus erythmatous (SLE), multiple sclerosis, Graves opthalmopathy, atopic rhinitis, atopic dermatitis, bullous phemphigoid, or other CD4+ cell mediated diseases, in animals, including humans and includes methods of treating such disorders. The compositions include at least one of the IL-16 antagonists, preferably at least the IL-16 peptide antagonist according to the present invention, admixed with a pharmaceutically acceptable carrier.
Nucleic acid molecules coding for any of the above IL-16 antagonist peptides of the present invention, expression vectors which include any of such nucleic acid molecules, as well as related host cells containing such nucleotide sequences or vectors, are also contemplated by the present invention.
Still another embodiment of the present invention is directed to antibodies raised against the IL-16 antagonist peptides of the present invention.
Preferably, the antibodies of the present invention are raised against those IL-16 antagonist peptides whose sequences coincide with the corresponding sequences of a mammalian IL-16 protein, which antibodies can antagonize or neutralize the activity of IL-16. Both polyclonal antibodies and monoclonal antibodies are contemplated by the present invention.
These and other embodiments of the invention will be readily apparent to those of ordinary skill in view of the disclosure herein.